Project Summary The incidence of infertility has increased 4% since the 1980s, with up to 20% cases having no known cause. One of the prevailing hypotheses is incompatibility between cognate egg and sperm proteins; however, very few pairs of interacting reproductive proteins have been identified in any organism. One of the best models for studying core mechanisms of fertilization is the marine gastropod abalone, where sperm are highly enriched for only a few proteins. The first identified pair of interacting reproductive proteins were abalone sperm lysin and its egg coat receptor VERL. As a major component of the abalone egg coat, VERL is a giant, fibrous glycoprotein composed of ~22 ZP-N repeats that likely form intermolecular hydrogen bonds to create the highly stabilized and protective egg coat. Lysin creates a hole in the egg coat by competing for these hydrogen bonds, allowing sperm to pass and fuse with the oocyte. These ZP-N repeats are also a principal component of mammalian egg coats, and multiple lines of evidence support the human protein ZP2 as the functional analog of VERL. However, three major outstanding questions are (1) besides lysin and VERL, which sperm and egg proteins are interacting, (2) what sites in these proteins are important for mediating this interaction, and (3) how might mutations in these sites impact protein structure and fertility. To address these fundamental questions of reproduction in both abalone (K99) and humans (R00), two specific aims are proposed that utilize state-of-the- art genomic, proteomic, and structural methods. In aim 1, for the K99 phase, I will combine long read sequencing technology, tests for molecular evolution, and quantitative proteomics to identify potential pairs of interacting reproductive proteins in abalone; for the R00 phase, I will extend these quantitative proteomic methods to identify potentially novel pathways that contribute to male infertility. In aim 2, for the K99 phase, the solution structure of VERL repeat 1 and its interactions with lysin will be determined by NMR, with deep mutational scanning used to identify mutations that interfere lysin-VERL interactions. In the R00 phase, these technologies will be applied to human ZP2 and I will identify mutations that interfere with sperm binding and characterize their structural effects. The proposed research is innovative for its combined use of genomic, proteomic, and structural techniques to characterize the molecular mechanisms underlying the interactions of rapidly evolving reproductive proteins. The results are expected to shed insight into the core processes that mediate egg-sperm interactions, and provide foundational information towards understanding the more complex mammalian system.